A novel γD-crystallin mutation causes mild changes in protein properties but leads to congenital coralliform cataract

Purpose To identify the genetic lesions for congenital coralliform cataract. Methods Two Chinese families with autosomal dominant coralliform cataract, 12 affected and 14 unaffected individuals, were recruited. Fifteen known genes associated with autosomal dominant congenital cataract were screened by two-point linkage analysis with gene based single nucleotide polymorphisms and microsatellite markers. Sequence variations were identified. Recombinant FLAG-tagged wild type or mutant γD-crystallin was expressed in human lens epithelial cells and COS-7 cells. Protein solubility and intracellular distribution were analyzed by western blotting and immunofluorescence, respectively. Results A novel heterozygous change, c.43C>A (R15S) of γD-crystallin (CRYGD) co-segregated with coralliform cataract in one family and a known substitution, c.70C>A (P24T), in the other family. Unaffected family members and 103 unrelated control subjects did not carry these mutations. Similar to the wild type protein, R15S γD-crystallin was detergent soluble and was located in the cytoplasm. ProtScale and ScanProsite analyses revealed raised local hydrophobicity and the creation of a hypothetical casein kinase II phosphorylation site. Conclusions A novel R15S mutation caused congenital coralliform cataract in a Chinese family. R15S possessed similar properties to the wild type γD-crystallin, but its predicted increase of hydrophobicity and putative phosphorylation site could lead to protein aggregation, subsequently causing opacification in lens.

CRYGD is a structural protein essential for lens transparency. Mutations of CRYGD are common genetic lesions causing different types of congenital cataracts. Among the reported families with congenital cataract caused by mutations of crystallin, one-third of them were associated with CRYGD. Until now, a total of 11 cataract-causing mutations (UniProt) have been reported including R15C, P24S, P24T, R37S, R59H, G61C, E107A, Y134X, W156X, G165fsX8, and R168W [17][18][19][20][21]. In this study, we added a novel R15S mutation to this list, affirming a causative role of CRYGD in coralliform type of congenital cataract.

METHODS
Affiliated Hospital (College of Medicine at Zhejiang University; Family B; Figure 1). A total of 26 family members including 12 affected and 14 unaffected individuals attended this study, and informed consents were obtained from all participants. Unrelated Chinese control subjects (n=103) attending the hospital clinics for ophthalmic examinations were also recruited. They did not have any eye diseases except senile cataract and mild floaters. All subjects underwent complete ophthalmoscopic examinations. Family history and ophthalmic examination were documented by senior ophthalmologists. Peripheral venous blood was collected for genomic DNA extraction using QIAamp DNA kit (Qiagen, Valencia, CA).
Candidate gene screening: Fifteen candidate genes that account for most cases of ADCC were taken for linkage analysis (Table 1). A gene exclusion strategy was conducted by screening with single nucleotide polymorphisms (SNPs) and microsatellite markers. We obtained gene-based SNP markers in Han Chinese from HapMap with ABI SNP browser v.3.5 (Applied Biosystems, Foster, CA). TaqMan SNP genotyping assay and allelic discrimination was conducted on an ABI PRISM 7000 sequence detection system (Applied Biosystems). For genes without informative SNP markers or those that could not be excluded by SNP linkage analysis, microsatellite markers flanking to the target genes were chosen from the Marshfield genetic map. GeneScan was conducted on ABIPRISM ® 377 DNA sequencer (Applied Biosystems). The pedigree and genotyping data were managed by GenoPedigree 1.0 and GeneBase 2.0.1. (Applied Biosystems). Two point LOD scores were calculated by the MLINK subprogram of FASTLINKAGE v.4.1P. A gene frequency of 0.0001 and penetrance of 100% were assumed for ADCC.
Computational analysis: Effects of amino acid changes on the CRYGD protein structure, the isoelectric point (pI), and molecular weight (MW) were examined by Expasy proteomics. Local hydrophobicity was predicted by ProtScale. The protein sequence was scanned by ScanProsite to predict the effect of the mutation on specific motifs. Figure 1. Pedigree of coralliform cataract families. The asterisk indicates family members who attended this study. The lens photograph from II-13 in Family A showed a line-shape opacity with a larger white dot end and tubular opacity radiating from the center of the lens. The lens photograph from II-2 in Family B showed a tubular and irregular opacity extending from center of the lens. Large white dots accumulated at the end of tubular protrusions.  Figure 1) and the Department of Ophthalmology at the First CRYGD expression, mutagenesis, and transfection: Human full-length wild type CRYGD was cloned to p3XFLAG-myc-CMV ™ -25 (Sigma, St Louis, MO) for epitope tagging to generate pFLAG/myc-CRYGD WT [19]. Missense mutations were introduced by a site-directed mutagenesis kit (Stratagene, La Jolla, CA) with specific oligonucleotides ( Table 3). Correctness of the construct sequence was confirmed by direct sequencing. Preparation of pFLAG-CRYGD G165fs was described as before [19]. Human lens B3 epithelial cells and COS-7 cells (ATCC, Manassas, VA) were maintained in Eagle's minimum essential medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum and antibiotics [19]. Cells were seeded with 5×10 5 cells in a 60 mm (diameter) culture dish (Nunc,

Gene Gene ID Chromosome location SNPs STRs
Gene name, gene ID in NCBI GeneBank , and the chromosomal location for 15 candidate genes were shown in this

Gene Amplicon Primer sequences
5′-CCTTTACGCTGCGTTTGATC 5′-AACCCCCAGACAAGAGGC Five genes which were sequenced were listed. Forward and reverse primer sequences were provided for each amplicon of each gene.

Mutations
Oligonucleotides with specific base change (underlined) 5′-TACTTCCTGCGCCGCTGCGACTATGCCGAC Constructs of four mutations in CRYGD were made. The sense oligonucleotides used in site-directed mutagenesis experiment were listed, respectively.
for 2 min on ice [22]. After centrifugation, the Tx-soluble fraction was collected and denatured in SDS buffer containing 50 mM DTT. The pellet containing Tx-insoluble proteins was sonicated and denatured in SDS buffer containing 9 M urea. Both Tx-soluble and Tx-insoluble proteins equivalent to 7.5×10 4 cells were analyzed by western blotting using monoclonal antibodies against FLAG, Glyceraldehyde 3phosphate dehydrogenase (GAPDH), or β-actin (Sigma).
Immunofluorescence staining: Cells grown on glass coverslips were fixed with freshly prepared neutral buffered 2% paraformaldehyde (Sigma) and permeabilized with 0.05% Tx [22] followed by incubations with mouse monoclonal antibodies against FLAG or mouse monoclonal antibodies against CRYGD (Abnova, Heidelberg, Germany). Rhodamine Red-X goat anti-mouse IgG (Invitrogen) was applied as the secondary antibody. The nuclei were contraststained with 4',6-diamidino-2-phenylindole (DAPI). The cells were examined under fluorescence microscopy (DMRB, Leica, Germany), which was equipped with Spot RT color system (Diagnostic Instruments Inc., Sterling Heights, MI).

Clinical investigations:
Two pedigrees exhibited coralliform type of cataract in an autosomal dominant mode of inheritance. All affected patients had bilateral lens opacification, characterized by the appearance of white lines and processes extending from the nucleus to peripheral cortex, resembling the shape of sea coral (Figure 1). This was classified as the coralliform type of cataract by senior ophthalmologists in two eye centers (D.S.P.F., D.L., and J.P.T.). The lens opacity was less severe in terms of size and density in Family A than in Family B and did not result in significant loss of visual acuity (VA). In family A, except for one patient, II10, who had low vision due to high myopia, VA of other patients ranged from 0.67 to 0.8 without lens surgery. Patient III-10 was recorded to have normal lens transparency at his first eye examination at the age of 2.5 years, but he was diagnosed to have cataract at 10 years old. Other family members were diagnosed after the age of five years. In Family B, all patients showed cataract within the first year after birth. The lens opacity caused obvious vision loss ranging from 0.04 to 0.7. Four of the six patients received cataract surgeries before the age of 30. Linkage analysis and DNA sequencing: Through linkage analysis with selected SNPs and microsatellite markers (LOD score equal to minus infinity), 10 ADCC-associated genes were excluded. Five other genes were subject to direct sequencing. These genes were CRYAA (D21S1255; LOD score 0.3), CRYAB (D11S4151; LOD score 0.9), CRYGC, CRYGD (D2S2361; LOD score 1.51), and MAF (D16S503; LOD score 0.9) in Family A and CRYAB (D11S1793; LOD score 2.41), CRYGC, and CRYGD (D2S2208; LOD score 2.41) in Family B. Two sequence variants in exon 2 of CRYGD (NCBI accession number NM_006891.3) were identified to segregate with cataract in these two families. In Family A, a novel missense transversion, c.43C>A, which led to a substitution of arginine with serine at the 15th amino acid position (R15S), was detected ( Figure 2). In Family B, a reported missense change, c.70C>A, which substituted proline with threonine at the 24th amino acid position (P24T), was found. All patients showed heterozygous changes. Neither the normal family members nor the 103 unrelated healthy controls carried these changes. Computational protein analysis of R15S CRYGD: By Expasy proteomics, R15S CRYGD was predicted to have a reduced isoelectric point (pI) of 6.58 (compared to 7.0 for the wild type). The molecular weight (MW) was also slightly decreased (20.669 kDa for R15S CRYGD versus 20.738 kDa for wild type). By ProtScale analysis, the local hydrophobicity at and near the altered amino acid was increased (Figure 3). By ScanProsite, a hypothetical casein kinase II phosphorylation site was created due to the R15S mutation (Table 4).
Cell specificity of R15S CRYGD solubility: Recombinant FLAG/myc-tagged wild type or cataract-causing mutant CRYGD (R15S, R15C, P24T, G61C, and G165fsX8) was expressed in human lens epithelial B3 cells and COS-7 cells. Tx-soluble and Tx-insoluble fractions were western blotted for FLAG to detect wild type and mutant CRYGD proteins. The result of expression in lens B3 cells showed that except for G165fsX8, all known mutants of CRYGD remained Txsoluble, which is similar to the wild type protein (Figure 4). The majority of G165fsX8 was present as Tx-insoluble. However, when expressed in COS-7 cells, an appreciable amount of mutant CRYGD protein became Tx-insoluble ( Figure 4) and wild type CRYGD remained Tx-soluble. Band densitometry followed by normalization with housekeeping proteins (GAPDH for Tx-soluble and β-actin for Tx-insoluble fractions) revealed that about 8% of R15C and 17% of R15S CRYGD were Tx-insoluble (compared to 0.2% of wild type CRYGD). Similar observations were obtained in triplicate experiments.
Cellular distribution: In COS-7 cells, recombinant FLAG/ myc-tagged R15S CRYGD was located predominantly in the cytoplasm and slightly in the nuclei (data not shown). No obvious inclusion was observed. This was similar to that observed in cells expressing wild type CRYGD.

DISCUSSION
In this study, we identified a novel R15S mutation and a P24T change of CRYGD in two Chinese families showing ADCC of the coralliform type, which is a rare subtype of bilateral Figure 4. Detergent solubility assay of FLAG-tagged wild type and mutant CRYGD. Western blotting analysis showed cell type specific solubility changes of CRYGD mutants. When expressed in human lens B3 epithelial cells (upper set of blots), wild type (WT) and all known mutant CRYGD (including R15S), except G165fsX8, were completely soluble in 0.5% Triton X-100 extraction. G165fsX8 was mainly Tx-insoluble. When expressed in COS-7 cells (lower set of blots), R15C and R15S CRYGD became moderately Tx-insoluble, unlike the wild type and P24T and G61C mutants. G165fsX8 remained Tx-insoluble. static congenital cataracts characterized by the appearance of opacity resembling the shape of sea coral. It shows white or cerulean opacification arranged in fusiform or spindle shape, extending from the center of the lens to the periphery but never reaching the capsule [10,11]. The irregular pattern of opacity running across the anatomic boundary of the cortical region indicates an altered arrangement of lens fibers, which associates with light scattering and reduction of transparency [11]. Until now, autosomal dominant transmission was the only mode of inheritance reported for coralliform cataract. Two loci, 2p24-pter and 2q33-35, have been mapped, and the specific gene, CRYGD (UniProt), in the latter locus was identified to associate with this cataract subtype [23]. Among all CRYGD mutations, R15C, P24T, and G61C were reported to be responsible for the coralliform phenotype [21,[24][25][26][27]. In this study, the novel R15S change in CRYGD was found to cause congenital coralliform cataract. The index patient, III-10, in in family A did not have a detectable lens opacity when he was 2.5 years old but was diagnosed with cataract at nine years of age. This was different from the effects of a previously reported R15C mutation of CRYGD, which caused the punctate type of congenital cataract at a much earlier age of disease onset [28]. Human CRYGD exists as a monomeric protein with a highly symmetric structure containing four Greek key motifs organized into two highly homologous βsheet domains. The NH2-terminal and COOH-terminal domains are covalently connected by a six-residue linker and interact non-covalently through the side chains of 10 amino acids across the domain interface. Due to these two conserved regions and a central hydrophobic domain interface, CRYGD exhibits high intrinsic stability [29,30]. For the R15C mutant, the additional reactive cysteine molecule at the protein surface could lead to the formation of disulfide cross-linkage, which in turn causes protein aggregation [31]. Nevertheless, substitution of the highly polar and charged arginine molecule by a less polar serine molecule as in R15S may cause lesser effects on protein conformation than the formation of an additional disulfide bond due to cysteine. Therefore, the R15S mutant is associated with the less severe late onset of cataract phenotype in our studied family. Although we detected no alteration of biochemical properties due to R15S in our cell expression studies, increased hydrophobicity at the R15S mutation site as predicted by ProtScale might affect proteinprotein interactions since R15 is located on a solvent accessible protein surface [28]. This would contribute to abnormal protein packing in the lens and would result in aggregate formation and thus lens opacification. Moreover, examination by ScanProsite predicted the creation of a putative casein kinase II phosphorylation site due to R15S. Whether this posttranslational modification is associated with protein interactions or oligomerization remains to be investigated. Additionally, an animal study had shown that the mutant γ-crystallin could cause cataract by the deposition of the misfiled protein as amyloid-like inclusions [32]. But this only explained those mutations which could affect protein structure severely, such as a frameshift mutation leading to protein truncation, ultimately causing mutant protein aggregate into inclusion. R15S was not supposed to have such a severe influence on protein structure. No obvious inclusion was observed in cells expressing R15S in this study. The P24T mutant of CRYGD is associated with diverse opacity morphology [24,25,[33][34][35]. The P24T and P24S mutants [18] are responsible for different crystallin cataracts such as the coralliform, aculeiform, fasciculiform, and crystal forms. In vitro expression of P24T CRYGD demonstrated reduced solubility when compared to the wild type protein [36,37]. The additional formation of a hydrogen bond might modify β-strand conformation, leading to protein insolubility rather than loss of stability. This might result in protein aggregation and then opacification in the lens.
In conclusion, a novel R15S mutation and a P24T mutation of CRYGD were identified in two Chinese families showing the coralliform type of congenital cataract. Different phenotypic features and onset time of disease in the two families accentuated the unique role of a single amino acid change in protein properties, which is crucial for disease pathogenesis. The enhanced hydrophobicity and a hypothetical phosphorylation site in the vicinity of R15S may affect the CRYGD protein interaction and the formation of the protein cluster.